GSM6809634: CD45p.coculture.CACO-2.untransf.noheat#1; Homo sapiens; RNA-Seq
We established a co-culture model in which ex vivo blood immunocytes were placed in direct contact with virus-infected epithelial cells or SARS-CoV2 single protein transfected epithelial cells. We used as a virus SARS-CoV2 and two other relevant viruses for comparison : influenza A virus and Ebola virus. Thirty-five hours after viral infection of the Caco-2 monolayer, unbound virus was removed and peripheral blood mononuclear cells (PBMC) from healthy donors (HD) were added to the cultures. These were harvested 14 h later, and subpopulations (epithelial cells, CD45+CD19+ and CD45+CD14+) were magnetically purified for transcriptome profiling by RNAseq. We used the same coculture model to assess the role of individual SARS-CoV2 proteins : Caco-2 cells were transfected, in biological duplicates, with a panel of 27 plasmids encoding single viral proteins or GFP as a control; Forty-eight hour later, these transfectants were co-cultured with HD PBMCs, and the monocytes as well as the transfected epithelial cells were profiled by RNAseq after 14 h. total RNA was captured and purified on RNAClean XP beads (Beckman Coulter). Polyadenylated mRNA was then selected using an anchored oligo(dT) primer (5′–AAGCAGTGGTATCAACGCAGAGTACT30VN-3) and converted to cDNA via reverse transcription. First-strand cDNA was subjected to limited PCR amplification followed by Tn5 transposon-based fragmentation using the Nextera XT DNA Library Preparation Kit (Illumina). Samples were then PCR amplified for 18 cycles using barcoded primers such that each sample carried a specific combination of eight-base Illumina P5 and P7 barcodes and were pooled together prior to Smart sequencing. Smart-seq paired-end sequencing was performed on an Illumina NextSeq500 using 2 x 38bp reads with no further trimming.